1. Field of the Invention
The present invention relates to a metal ion adsorbent.
2. Prior Art
It is known that by introducing a chelate forming group into a polymer, such as, polystyrene, there is obtained an adsorbent for bivalent or multivalent metal ions. Such a chelate adsorbent usually binds the metal ion stronger and more selectively than the usual, non-chelating ion exchangers.
It is furthermore known that a cross linked polymer with chelate forming groups which are charged with metal ions (such as bivalent copper or zinc) could be used for adsorption of substances which coordinate with these metal ions. Chromatography based on this principle has been denoted "ligand exchange chromatography" (F. Helfferich, J. Amer. Chem. Soc. 84 (1962) 3242). Ligands such as water, buffer ions, etc., could be exchanged for the adsorbing substances.
Also high molecular substances, such as proteins could be adsorbed to the metal ions fixed in the matrix. This embodiment of the chromatographic procedure has been denoted metal chelate affinity chromatography (J. Porath et al., Nature 258 (1975)598).
The phenomenon metal chelate adsorption could also be used in batch procedures where a solution containing substances having an affinity to the polymer attached metal is brought in contact with the metal (i.e., suspended in the solution). After the adsorption the gel is removed and washed whereafter material adsorbed can be diluted by means of changing the composition of the medium (change of pH, change of salt or by means of introduction of a chelating substance). The metal chelate adsorption could also be used for immobilizing proteins, such as enzymes, antibodies and antigens.
In adsorption-desorption procedures including biopolymers such as protein or nucleic acids, an hydrophilic matrix has been used to which a chelate forming group has been bound covalently. It is important that the gel matrix is hydrophilic as one would otherwise obtain a strong mutual effect between the protein and the matrix substance in a water solution ("hydrofob adsorption"). The selectiveness and specific character of the adsorption is not achieved in strong hydrofob adsorption and the product will not be useful for protein- and nucleic acid purification.
In order to obtain an optimal function the metal ion should be bound so strongly to the carrying polymer matrix that it is not desorbed (released) under the conditions useful for carrying out the adsorption-desorption procedures in a batch or at chromatography. Usually, these procedures are performed in the temperature range of 0.degree.-40.degree. and within a pH-range of pH 3-10. The metal ion shall not be removed from the polymer by substances which are normally contained in the solutions or extracts which are subject to the process. The extracts from organisms often contain ammonium, amines, amino acids or other metal coordinating substances. The metal is not removed by these substances from an ideal chelate adsorber.
On the other hand, the metal ion has to have a certain residual affinity so that proteins and the above mentioned substances can coordinate with the metal (without removing the metal from the polymer attached ligands). Metal ions like zinc, iron, cobalt, etc., usually coodinate with 6 ligands. If all of the ligands are fixed into the matrix there is no residual affinity for ligands containing substances of a lower metal affinity than that of the polymer fixating ligands in the surrounding solution--the metal ions are coordinately saturated with strong ligands. If, however, many "free" coordination positions are available the metal ion often is too loosely bound to the polymer and is removed from the gel simultaneously with the adsorbed protein at the desorption. Furthermore, the adsorbent is less well defined considering the adsorption properties of the metal chelate group. Different metal chelate groups get different contents of the ligands adsorbed from the solution (due to different steric surrounding).
The group hitherto normally used is the iminodiacetate group: ##STR1## This group together with a copper ion forms the following chelate: ##STR2## This group gives the copper three polymer bound and three free ligands.
The following group has also been used with Sephadex as a carrying polymer matrix: ##STR3## (see J. Porath, J. Chromatogr 159 (1978) p. 23).
Here 7 metal coordinating atoms are present: 2 N and 5 O from the carboxylic groups. In accordance with the discussions above this number of ligands is unsuitably high. A group according to this formula could form a metal ion for instance Cu.sup.2+ (a), but when this is the case a further metal ion can be formed and this second metal ion (b) is: ##STR4## The difference is obvious. The metal ion (a) is more strongly bound to the adsorbing element than the metal ion (b). On the other hand, four free coordinating positions are available at (b) but only one at (a) which implies that (b) more strongly ties the ligand substances in a solution. An adsorbing means having so many ligands atoms for each metal binding group will therefore be relatively heterogeneous.